1. Field
This disclosure is concerned generally with protein purification and specifically with a method of purifying .alpha.-1 proteinase inhibitor from plasma fractions with cation exchange under conditions such that active .alpha.-1 PI does not bind to the column but contaminating proteins do.
2. Prior Art
Alpha-1 proteinase inhibitor (.alpha.-1 PI) is a glycoprotein with a molecular weight of about 55,000 Daltons. Alpha-1 PI is an inhibitor of proteases such as trypsin, chymotrypsin, pancreatic elastase, skin collagenase, renin, urokinase and proteases of polymorphonuclear lymphocytes. A current therapeutic use of .alpha.-1 PI is the inhibition of lymphocyte elastase in the lungs. This protease functions by breaking down foreign proteins. When .alpha.-1 PI is not present in sufficient quantities to regulate elastase activity, the elastase breaks down lung tissue. In time this imbalance results in chronic lung tissue damage and emphysema. Alpha-1 PI replenishment has been successfully used for treatment of this form of emphysema.
Currently the demand for .alpha.-1 PI exceeds the available supply. The .alpha.-1 PI gene has been transferred and expressed in microorganisms, cell lines and sheep. However, a satisfactory recombinant product has yet to be produced. Human plasma is still the only approved source of therapeutic .alpha.-1 PI. Alpha-1 PI is used for replacement therapy and is given to patients on a regular basis over extended periods of time. Because trace impurities can stimulate an immune response in patients, high purity of the product is critical to successful treatment. Plasma, the source of .alpha.-1 PI, is limited and therefore a purification process with high yield of .alpha.-1 PI is necessary. To date a practical process which gives both high yield and high purity .alpha.-1 PI has not been available.
Various methods of purifying .alpha.-1 PI from human plasma have been described. Bollen et al., U.S. Pat. No. 4,629,567 (1986) used five different chromatography steps to purify the .alpha.-1 PI from yeast, E. coli and human plasma. The five steps involved DEAE ion exchange, thiol-disulfide exchange, heparin affinity, zinc-chelate chromatography, and amino hexyl ion exchange. No purity and yield data were shown.
Novika et al., Gematol. Transfuziol. 34:46-50(1989) reported isolation methods from the by-products of the manufacture of blood products. They used affinity, DEAE cellulose, and gel filtration chromatographies. The purity and yield data were not available.
Podiarene et al., Vopr. Med. Khim. 35:96-99(1989) reported a single step procedure for isolation of .alpha.-1 PI from human plasma using affinity chromatography with monoclonal antibodies. Alpha-1 PI activity was increased 61.1 fold with a yield of 20%.
Burnouf et al., Vox. Sang. 52, 291-297(1987) starting with plasma supernatant A (equivalent to Cohn Fraction II+III) used DEAE chromatography and size exclusion chromatography to produce an .alpha.-1 PI which was 80-90% pure (by SDS-PAGE) with a 36-fold increase in purity. Recovery was 65-70% from the supernatant A.
Hein et al., Eur. Respir. J. 9:16s-20s (1990) presented a process which employs Cohn Fraction IV-1 as the starting material and utilized fractional precipitation with polyethylene glycol followed by anion exchange chromatography on DEAE Sepharose.RTM.. The final product has a purity of about 60% with 45% yield.
Dubin et al., Prep. Biochem. 20:63-70 (1990) have shown a two step chromatographic purification. First .alpha.-1 PI, CI inhibitor, .alpha.-1 antichymotrypsin, and inter .alpha.-1 trypsin inhibitor were eluted from Blue Sepharose.RTM. and then .alpha.-1 PI was purified by gel filtration. Purity and yield data were not available.
Ballieux et al. purified an .alpha.-1 PI and proteinase-3 complex from purulent sputum using 4-phenylbutylamine affinity chromatography, cation exchange, and a final immunoaffinity step (Ballieux, B. E. et al., J. Immunol. Methods 159:63-70 (1993)). The pH of the buffer used in the cation exchange step was 7.0. Under the conditions used, most of the sputum proteins bound to the resin but .alpha.-1 PI and proteinase-3 passed through without binding.
Jordan et al., U.S. Pat. No. 4,749,783 (1988) described a method where biologically inactive proteins in a preparation were removed by affinity chromatography after a viral inactivation step. The basis of the separation between the native and denatured forms of the protein was the biological activity of the native protein towards the affinity resin and not physical differences between the native and denatured proteins.
None of these processes have used flow through chromatography with strong cation resins at low pH, low salt concentration and moderate protein concentration as a purification step. Unexpectedly, under conditions such as these, only active .alpha.-1 PI flows through the column. The process can be arranged to achieve 90% yield from the chromatography column and around 95% purity after 2 applications of the cation exchange column. The present invention provides an improved process for purification of .alpha.-1 PI from human plasma at large scale with both high purity and high yield.